Receptor subtype and function selective retinoid and rexinoid compounds in combination with immune modulators for cancer immunotherapy

ABSTRACT

Disclosed herein are methods for culturing CAR-modified immune cells with at least one Retinoic Acid Receptor and/or Retinoid X Receptor active agent.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/281,402, filed Nov. 26, 2019, now U.S. Pat. No. 10,485,775, which isa divisional of U.S. patent application Ser. No. 16/034,064, filed Jul.12, 2018, now U.S. Pat. No. 10,231,944, which claims the benefit of U.S.Provisional Patent Applications No. 62/532,233, filed on Jul. 13, 2017and 62/552,814, filed on Aug. 31, 2017; the entire contents of which areeach incorporated by reference herein.

BACKGROUND

For years, the cornerstones of cancer treatment have been surgery,chemotherapy, and radiation therapy. Over the last decade, targetedtherapies—drugs that target cancer cells by homing in on specificmolecular changes seen primarily in those cells—have also emerged asstandard treatments for a number of cancers. One approach toimmunotherapy involves engineering immune cells to recognize and attacktumors.

SUMMARY

Disclosed herein are compounds for potentiation of targeted cancerimmunotherapeutics. Compounds which act on retinoic acid receptors (RAR)and retinoid X receptors (RXR) are used in combination with chimericantigen receptor (CAR)-modified immune cells (sometimes abbreviated asCAR-MIC) to potentiate the anti-cancer activity.

Thus, provided herein are methods of treating cancer, the methodscomprising administering CAR-modified immune cells and at least oneretinoid active agent and/or rexinoid active agent (collectively RAR/RXRactive agents). In some embodiments, the retinoid active agent is aRetinoic Acid Receptor (RAR) active agent. In some embodiments, therexinoid active agent is a Retinoid X Receptor (RXR) active agent. Insome embodiments, two RAR/RXR active agents are used; they can be twoRAR active agents, two RXR active agents, or a RAR active agent and aRXR active agent. In some embodiments the RAR/RXR active agent acts asan agonist of its receptor while in other embodiments the RAR/RXR activeagent acts as an antagonist of its receptor. In some embodimentsutilizing multiple RAR/RXR active agents, the multiple RAR/RXR activeagents are formulated and administered separately. In some aspects ofthese embodiments, the RAR/RXR active agents are administeredseparately, but during the same treatment session. In other aspects ofthese embodiments, the RAR/RXR active agents are administered indifferent treatment sessions. In other embodiments, the multiple RAR/RXRactive agents are formulated separately, but co-administered (that is,administered during the same treatment session). In still otherembodiments, the multiple RAR/RXR active agents are formulated togetheras a single, common medicament.

In some embodiments, the CAR-modified immune cells are, or comprise,CAR-modified T cells. In some embodiments, the CAR-modified immune cellsare, or comprise, CAR-modified NK cells. In some embodiments, theCAR-modified immune cells are, or comprise, CAR-modified NKT cells. Insome embodiments, the CAR-modified immune cells are, or comprise,CAR-modified macrophages. Further embodiments can comprise mixtures ofthese cell types. Most typically such cellular preparations areadministered by infusion, for example intravenous infusion. In contrast,the RAR/RXR active agents are small molecules that can be administeredorally, for example as pills or capsules and the like. Thus the RAR/RXRactive agents and the CAR-modified immune cells may be administered onindependent schedules.

In some embodiments, the retinoid active agent is a RARα antagonist. Insome embodiments, the RARα antagonist is a compound of general formula(I):

wherein R¹, R², R³, and R⁶ are independently H or C₁₋₆ alkyl; R⁴ and R⁵are independently H or F; Ar is phenyl, pyridyl, thienyl, furyl, ornaphthyl; X is C(CH₃)₂, O, S, or NR⁷, wherein R⁷ is H or C₁₋₆ alkyl; X¹is H or halogen such as F, Cl or Br; and R⁸ is H or OH.

In some embodiments, the RARα antagonist is AGN194301, AGN193491,AGN193618, AGN194202, or AGN194574.

In some embodiments, the RARα antagonist is a compound of generalformula (II):

wherein R¹ and R² are independently C₁₋₆ alkyl; X is O, S, or CH₂; Y isO, S, CH₂, or NR³, wherein R³ is C₁₋₆ alkyl; Z is Cl or Br; W is H orOH; and U is independently H or F. In some embodiments, the RARαantagonist is:

In some embodiments, the RARα antagonist is a compound of generalformula (III):

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; Ar isphenyl, pyridyl, thienyl, furyl, or naphthyl; X is O, S, N, or CH₂; W isH or OH; and Z is Cl or Br.

In some embodiments, the RARα antagonist is:

In some embodiments, the RARα antagonist is

In some embodiments, the retinoid active agent is a RAR agonist. In someembodiments, the RAR agonist is:

In some embodiments, the RAR agonist is a RARy selective agonist ofgeneral formula (IV):

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; and Xis O, S, CH₂, C(R⁴)₂, or NR⁵, wherein R⁴ and R⁵ are independently H orC₁₋₆ alkyl.

In some embodiments, the RAR agonist is a RARγ selective agonistselected from

In some embodiments, the retinoid active agent is a RXR antagonist. Insome embodiments, the RXR antagonist is:

In some embodiments, the RXR antagonist is AGN195393, or LGN100849.

In some embodiments, the methods comprise additionally administering atleast one cancer chemotherapy agent.

In some embodiments, the methods comprise administering at least tworetinoid active agents. In some embodiments, the two retinoid activeagents are a RARα antagonist and a RARγ agonist.

In some embodiments, the methods further comprise administering to thesubject at least one immune checkpoint inhibitor. In some embodiments,the immune checkpoint inhibitor is an inhibitor of at least one ofCTLA-4, PD-1, TIM-3, LAG-3, PD-L1 ligand, B7-H3, B7-H4, BTLA, or is anICOS, or OX40 agonist. In some embodiments, the immune checkpointinhibitor is an antibody specific for at least one of CTLA-4, PD-1,TIM-3, LAG-3, PD-L1 ligand, B7-H3, B7-H4, BTLA, ICOS, or OX40.

Also disclosed herein are methods of prolonging the disease-freesurvival of a cancer patient comprising administering CAR-modifiedimmune cells and at least one retinoid active agent and/or rexinoidactive agent.

Also disclosed herein are methods of decreasing toxicity of CAR-modifiedimmune cells comprising administering to a subject in need thereof atleast one retinoid active agent and/or rexinoid active agent incombination with the CAR-modified immune cells such that as a result ofthe combination, a lower dose of CAR-modified immune cells areadministered more safely and equally effectively than if theCAR-modified immune cells were administered alone; or that a higher doseof CAR-MIC can be administered with greater efficacy and equal safety.

Also disclosed herein are methods of expanding the number ofCAR-modified immune cells comprising culturing the CAR-modified immunecells in a culture medium comprising at least one retinoid active agentand/or rexinoid active agent. In some embodiments this is done insteadof administering RAR/RXR active agent(s) to the patient. In otherembodiments this is done in addition to administering RAR/RXR activeagent(s) to the patient. In various embodiments the RAR/RXR activeagent(s) used in the CAR-modified immune cell culture and thoseadministered to the patient are different, the same, or one setconstitutes a subset of the other.

Also disclosed herein are methods of treating cancer comprisingadministering to a subject in need thereof, chimeric antigen receptor(CAR)-modified immune cells, at least one retinoid active agent and/orrexinoid active agent, and at least one immune checkpoint inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C shows that RAR receptor specific agonists regulate FoxP3,a4β7, and CCR9 expression. Purified CD4⁺ CD25⁻ FoxP3⁻ cells werecultured in media with the specified concentration of each RAR agonistand analyzed by flow cytometry for FoxP3 (FIG. 1A), a4β7 (FIG. 1B), andCCR9 (FIG. 1C) expression in total CD4 T cells. FoxP3 results arerepresentative of 3 independent experiments. CCR9 and a4β7 results arerepresentative of multiple experiments.

DETAILED DESCRIPTION

Disclosed herein are combinations for therapy of cancer comprisingretinoid and/or rexinoid compounds and adoptive transfer of immune cellsexpressing chimeric antigen receptors (CAR-modified immune cells orCAR-MIC). Compounds which act on retinoic acid receptors (RAR) and/orretinoid X receptors (RXR) augment the activity of CAR-modified immunecells. By potentiation it is meant that the CAR-modified immune cellshave greater and/or more rapid effect when the RAR/RXR active agent isused with the CAR-modified immune cells than when the RAR/RXR activeagent is not used with the CAR-modified immune cells or, similarly, thata given degree of effect can be obtained with a smaller dosage ofCAR-modified immune cells when the RAR/RXR active agent is also usedthan would be required if the RAR/RXR active agent were not used.

As used herein, the term “potentiate” refers to an improved efficacy ofCAR-modified immune cells, or improved response by the patient, whenused in combination with a RAR/RXR active agent—especially an RARαantagonist, an RARγ agonist, an RXR antagonist, or combinationsthereof—compared to the use of CAR-modified immune cells in the absenceof the RAR/RXR active agent(s). As used herein, the term “augment” alsorefers to an improved effect when using an RAR/RXR active agent whencompared to the situation where the RAR/RXR active agent is not used.The potentiation described herein arises from theimmunoregulatory/immunomodulatory activity of the RAR/RXR activeagent(s).

Multiple modes of potentiation are possible. In some modes the RAR/RXRactive agent(s) acts directly on the CAR-modified immune cells. Asdelineated below, this can involve increasing the number or potency ofeffector cells and/or the suppression of Treg cells depending on theparticular RAR/RXR active agent(s) used. These effects can be obtainedby including the RAR/RXR active agent(s) in the preparatory cultures ofthe CAR-modified immune cells or by administering the RAR/RXR activeagent(s) to the patient along with and/or subsequent to administrationof the CAR-modified immune cells. In some modes the RAR/RXR activeagent(s) act in conjunction with the CAR-modified immune cells by 1)modifying the tumor environment by reducing the presence or activity ofTreg cells in the tumor thereby making the tumor more susceptible toimmunologic attack, and/or 2) generating a pro-inflammatory responsethat acts on the CAR-modified immune cells to promote theireffectiveness. These effects are generally dependent on the RAR/RXRactive agent(s) being administered to the patient. Additionally, ageneral antitumor immune response in the patient promoted by RAR/RXRactive agent(s) may further increase the overall effectiveness of thesetreatments.

Retinoic acid (RA), at higher pharmacological concentrations, causesanti-inflammatory effects by increasing levels of suppressive CD4⁺regulatory T cells (Treg cells). RA affects this function by enhancingexpression of the transcription factor Fox P3 which is the masterregulator of Treg cell differentiation. RA also reduces the levels ofpro-inflammatory Th17 cells. RA elicits these effects by activating theRARα subtype of retinoic acid receptors. The above functions of RA orRARα selective agonists result in these compounds contributing toresistance of tumors to immunotherapy. The increased levels ofsuppressor Treg cells impede the anti-tumor activity of the T cellsproduced by immunotherapy. The complement of T cells attacking the tumoris also reduced by the RARα agonist since it reduces the levels of Th17cells. Conversely, an antagonist of RARα sensitizes tumors toimmunotherapy because the RARα antagonist reduces levels of thesuppressive Treg cells and also increase levels of the effector Th17cells. Thus, in one embodiment disclosed herein, a target cancer istreated with a combination of CAR-modified immune cells in combinationwith an RARα antagonist.

In another aspect of RA function, it has been shown that physiologicalconcentrations of RA are critical for the development of T cell mediatedimmune responses. RA signaling to T cells is a critical early mediatorof CD4⁺ T cell effector function. Using T cells expressing dominantnegative RARα (dnRARα), a modified RARα which abrogates RAR function, ora RAR antagonist, it was shown that RA signaling through RARα isrequired for T cell mediated functions such as skin graft rejection.Thus, in the context of cancer immunotherapy, use of RARα antagonists,or RARα inverse agonists, in combination with CAR-modified immune cellshas counteracting effects: it can promote anti-tumor effects bydecreasing levels of suppressive Treg cells, but such antagonists canalso reduce anti-tumor effects by blocking CD4⁺ T cell effectorfunction. In this context, the use of RARα antagonists in combinationwith cancer immunotherapy may be of limited value and may even bedetrimental.

In another embodiment disclosed herein, the RA signaling that iscritical for the anti-cancer immune response is mediated by RARγ. In theabove scenario, the sole use of RARα antagonists in conjunction withcancer immunotherapy will result only in a reduction of suppressor Tregcells and consequently in a limited enhancement of the anti-tumoreffects of the immunotherapy. However, that approach does not takeadvantage of the early effects of RA or other RAR agonists actingthrough RARγ on promoting CD4⁺ T cell effector function and thepotential substantial enhancement of anti-tumor effects ofco-administered cancer immunotherapy. Thus, RAR agonists which actspecifically through RARγ will promote CD4⁺ T cell effector functionwithout increasing Treg cells and such RARγ selective agonists willsubstantially enhance the anti-tumor effects of cancer immunotherapy. Inyet another embodiment, the cancer immunotherapy is used to treat atumor together with a combination of a RARα antagonist and a RARγagonist. In this situation, the retinoid compounds will enhance theanti-tumor activity of the immunotherapy by the following mechanisms:the RARγ agonist will facilitate the development of a robust CD4⁺ T cellmediated immune response; the RARα antagonist will reduce the level ofsuppressor Treg cells and maintain the level of Th17 cells therebyminimizing modulation of the anti-tumor effects of the immunotherapy. Itshould be understood that the effect of using a RARα antagonist and a(non-selective) RAR agonist will be similar to using RARα antagonist anda RARγ agonist as the RARα antagonist will block the RARα agonisticactivity of the (non-selective) RAR agonist.

RXR agonists promote the formation of suppressor Treg cells and inhibitthe formation of effector Th17 cells. Thus in other embodiments, the useof a RXR antagonist (or inverse agonist) in combination withCAR-modified immune cells will enhance anti-tumor activity by decreasingformation of suppressor Treg cells and by increasing levels of Th17effector cells.

In summary, the following classes of compounds will be useful incombination to increase the anti-tumor activity of cancer immunotherapy:RARα antagonists, RARγ agonists, and RXR antagonists. In the methodsdisclosed herein, CAR-modified immune cells are administered incombination with one or more of RAR/RXR active agents (for example, RARαantagonists, RARγ agonists, RXR antagonists), with or without otheragents to treat certain cancers. The properties of RARα antagonism andRARγ agonism maybe present together in the same molecule. Thus, the samemolecule acting as an antagonist at RARα can reduce Treg cell formationand, simultaneously, acting as an agonist at RARγ further reduce Tregcell formation and promote CD4⁺ T cell effector function. In the samemanner, the properties of RXR antagonism may be separately combined withthe properties of RARα antagonism or RARγ agonism in distinct molecules.As used herein, the term “retinoid active agents” encompasses, withoutlimitation, any compound acting on a RAR. Non-limiting examples ofretinoid active agents are RARα antagonists and RARγ agonists. As usedherein, the term “rexinoid active agents” encompasses, withoutlimitation, any compound acting on a RXR. A non-limiting example of arexinoid active agent is a RXR antagonist.

RAR/RXR active agents, as a class, and in many cases individually, arepleiotropic in effect. In the disclosed embodiments RAR/RXR activeagents (for example, RARα antagonists, RARγ agonists, RXR antagonists)are used as immunotherapeutics or immunotherapeutic potentiators. Thisis an indirect mechanism of action in that the crucial effect is uponcells of the immune system rather than directly upon tumor cells. Theseor other RAR/RXR active agents may have other effects that may be usefulin the treatment of some cancers by acting directly on the cancer cellseither through a RAR/RXR-mediated mechanism (for example RXRantagonists) or through a non-RAR/RXR-mediated mechanism.

Cancer therapy can proceed through many mechanisms. Some anti-canceragents are classified as anti-proliferative agents. These include thelong-established chemotherapeutic agents which are generally cytotoxicas well as the more recently developed targeted therapies, such askinase inhibitors which act upon growth regulating pathways in thecancer cells, and antibody-based therapeutics that recognizecell-surface antigens on the cancer cells. Other therapeutic modalitiesinclude anti-neovasculature, in which the in-growth of blood vesselsinto the tumor to supply it with nutrients is disrupted, andanti-hormonal in which hormone-dependent tumors are treated bydisrupting hormonal supply or signaling.

It is also possible to distinguish between various modes ofimmunotherapy. For example one can distinguish between antibody-basedtherapies and cell-based therapies, and between passive and activetherapies. As used herein passive therapy refers to a therapy in whichthe primary immunotherapeutic agent is administered to the patient. Asused herein an active therapy refers to a therapy in which the primaryimmunotherapeutic agent is a component of an immune response induced inthe patient by the administered agent, for example, a vaccine. Otherimmunotherapeutic agents are classified as immunomodulatory agents. Asused herein the primary activity immunomodulatory agents is not directtherapeutic effect on the target disease, but rather increases ordecreases the production or activity of immune system components thatmediate or promote therapeutic effect. Such components of the immunesystem (cells or antibodies) act directly on the antigenic target orotherwise respond to antigenic stimulus to promote such a response, thatis, in the currently disclosed embodiments, immune system componentsthat act directly on tumor cells, particularly cancer cells, or providehelper function. Thus, in embodiments comprising administration ofCAR-modified immune cells to a cancer patient, the CAR-modified immunecells are to be considered a passive, cellular immunotherapeutic. In afurther aspect of these embodiments the CAR-modified immune cells havedirect cytotoxic effect. In embodiments involving use of RAR/RXR activeagents, whether in CAR-modified immune cell culture or administered to acancer patient, the RAR/RXR active agents are to be consideredimmunomodulatory agents. Similarly, in those embodiments involvingadministration of an immune checkpoint inhibitor, the immune checkpointinhibitor is to be considered an immunomodulatory agent, even if theimmune checkpoint inhibitor is an antibody.

Various embodiments are directed exclusively to an immunotherapeuticmechanism, that is, the RAR/RXR active agents are used promote animmunological attack on the tumor, and other activities the RAR/RXRactive agents may possess, if any, are not crucial to effectiveness.Some embodiments may exclude agents possessing other anticanceractivities. Other embodiments may take advantage of additionalactivities of the RAR/RXR active agent(s). Similarly, some embodimentsentail administration of only the RAR/RXR active agent(s) and theCAR-modified immune cells. Other embodiments are permissive ofcombination with other therapies and therapeutic agents. Some of theseembodiments specifically include one or another of the other therapiesand therapeutic agents. Others or these embodiments specifically excludeone or another of the other therapies and therapeutic agents. Othertherapies or therapeutic agents include other immunotherapies,anti-proliferative therapy, chemotherapy, cytotoxic agents, cytostaticagents, targeted therapy, radiation therapy, anti-hormonal therapy,anti-neovasculature therapy, anti-tumor antigen antibodies, anti-cancervaccines, immune checkpoint inhibitors, and immune checkpoint inhibitorantibodies. Thus, for example, some embodiments specifically include orexclude use of immune checkpoint inhibitors, or permit combination withimmune checkpoint inhibitors, but exclude other immunotherapeutics orother cancer therapies.

The term “agonist” as used herein shall be understood to mean a compoundwhich binds to a receptor and activates it, producing gene transcriptionand a subsequent pharmacological response (e.g., contraction,relaxation, secretion, enzyme activation, etc.). As used herein, theterm “RARγ agonist” refers to a compound that binds to RARγ with ahigher affinity compared to binding with another molecule, such as adifferent RAR. In exemplary embodiments, a RARγ agonist is selective forRARγ over RARα and/or RARβ. Thus, a RAR selective agonist tends to bindto a particular RAR receptor target with high binding affinity. As usedherein, the term “agonist” includes selective agonists.

The term “antagonist” as used herein, refers to a compound thatattenuates the effect of an agonist by binding in the same site as anagonist without activating the receptor. An antagonist by itself willnot affect the gene transcriptional activity of the unoccupied receptor.Conventionally, a RARα antagonist is a chemical agent that inhibits theactivity of an RARα agonist. As used herein, the term “RXR antagonist”refers to compounds that bind to RXR and do not activate it, but insteadantagonize transcription produced by a RXR agonist. As used herein, theterm “antagonist” includes selective antagonists.

The term “inverse agonist” as used herein shall be understood to mean acompound which produces an effect opposite to that of an agonist, yetacts at the same receptor. An inverse agonist by itself will reduce thebasal gene transcriptional activity of the unoccupied receptor.

RARα Antagonists

In certain embodiments, the RARα selective antagonist is a compoundrepresented by the general formula (I):

wherein R¹, R², R³, and R⁶ are independently H or C₁₋₆ alkyl; R⁴ and R⁵are independently H or F; Ar is phenyl, pyridyl, thienyl, furyl, ornaphthyl; X is C(CH₃)₂, O, S, or NR⁷, wherein R⁷ is H or C₁₋₆ alkyl; X¹is H or halogen such as F, Cl or Br; and R⁸ is H or OH. Each combinationof R groups and each combination of their independently selectedsubstituents defines a distinct individual embodiment.

An exemplary RARα selective antagonist of the general formula (I) is thecompound AGN194301:

Other exemplary RARα antagonists of the general class of general formula(I) include, but are not limited to, AGN193491, AGN193618, AGN194202,AGN193625, and AGN194574.

In other embodiments, the RARα selective antagonist is a member of theclass of compounds represented by general formula (II)

wherein R¹ and R² are independently C₁₋₆ alkyl; X is O, S, or CH₂; Y isO, S, CH₂, or NR³, wherein R³ is C₁₋₆ alkyl; Z is Cl or Br; W is H orOH; and U is independently H or F. Each combination of R groups and eachcombination of their independently selected substituents defines adistinct individual embodiment.

An exemplary RARα selective antagonist of the class represented bygeneral formula (II) for use in the methods disclosed herein isrepresented by the following structure (VTP196696):

In other embodiments, RARα selective antagonists are compounds of thegeneral formula (III).

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; Ar isphenyl, pyridyl, thienyl, furyl, or naphthyl; X is O, S, N, or CH₂; W isH or OH; and Z is Cl or Br. Each combination of R groups and eachcombination of their independently selected substituents defines adistinct individual embodiment.

An exemplary compound of general formula (III) is AGN194777.

Other exemplary RARα antagonists include, but are not limited to,BMS185411, BMS614, Ro41-5253, and Ro46-5471.

Additional RAR antagonists or inverse agonists are described in U.S.Pat. Nos. 6,037,488, 5,612,356, 5,776,699, 5,958,954, 5,877,207,6,225,494, 6,455,701, 5,723,666, 5,739,338, and 5,919,970, and US PatentApplication 2009/0176862, all of which are incorporated by referenceherein for all they disclose of RAR antagonists.

RARγ Agonists

Exemplary RARγ agonists are disclosed in U.S. Pat. Nos. 5,234,926,4,326,055, 5,324,840, 5,824,685, and 6,452,032, including but notlimited to the following compounds.

Another exemplary RARγ agonist is AGN 190168.

Although compounds such as AGN190183, AGN190205, AGN190168 (tazarotene)are RARγ agonists they are not RARγ selective since they activate RARαand/or RARβ as well. It may be preferable to use RARγ selective agonistssince activation of RARα may negate the T effector cell activationeffects produced by RARγ activation by increasing production of Tregcells. RARγ selective agonists, on the other hand, will potentiate theanti-tumor effects of cancer immunotherapeutics.

An example of a highly selective RARγ agonist is the compound:

Other RARγ selective agonists are members of the family of compounds ofgeneral formula (IV):

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; and Xis O, S, CH₂, C(R⁴)₂, or NR⁵, wherein R⁴ and R⁵ are independently H orC₁₋₆ alkyl. Each combination of R groups and each combination of theirindependently selected substituents defines a distinct individualembodiment.

Additional RARγ selective agonists include, but are not limited to,CD437, CD2325, CD666, and BMS961. Additional RARγ agonists are describedin International Publication WO 02/28810A2 which is incorporated byreference herein for all it discloses regarding RARγ agonists.

RXR Antagonists

Exemplary RXR antagonists include, but are not limited to, AGN195393,LGN100849, HX531, LG100754, PA451, PA452, and UVI 3003.

CAR-Modified Immune Cells

Tumor cells often down-regulate major histocompatibility complex (MHC)expression and furthermore, when they do express MHC alleles, theimmunodominant epitopes are not often known. Thus, MHC-dependent cancerimmunotherapies are often not effective. Chimeric antigen receptor(CAR)-modified immune cells react with target antigens on cancer cellsin an MHC-independent matter. The CAR allows binding via theantigen-binding domain to a target cells wherein the CAR-modified cellskill the target cells in a MHC non-restricted manner by binding to thetarget cells and induction of activation, proliferation, andcytotoxicity of the modified cells against the tumor target.

As used herein, the term “target cells” refers to cells expressing asurface antigen that can be bound by the CAR. The antigen can also bereferred to as the “target antigen.” Target antigens are antigens thatare differentially expressed on cancer cells such that the CAR targetsthe cancer cells preferentially over non-cancer cells.

Once the modified immune cells bind to target antigen, the internalstimulatory domains provide the necessary signals for the immune cell tobecome fully active. In this fully active state, the immune cells canmore effectively proliferate and attack cancer cells.

CAR-modified cells can recognize a variety of types of antigen, not onlyprotein but also carbohydrate and glycolipid structures typicallyexpressed on the tumor cell surface. Unlike T cell receptor (TCR)recognition, the antigen does not need to be processed and presented byMHC and therefore the same CAR-molecule can be used in all patients whoexpress the same tumor antigen regardless of HLA type.

The CAR comprises a recombinant polypeptide construct comprising atleast an antigen-binding domain, a transmembrane domain, and one or moreintracellular stimulatory domains (also referred to as a cytoplasmicsignaling domain or an intracellular signaling domain). Theantigen-binding domain allows the modified immune cells to specificallybind to the target tissue, the transmembrane domain anchor the CAR tothe immune cells, and the intracellular stimulatory domain inducespersistence, trafficking, and effector functions in the transducedcells.

The antigen-binding domain of a CAR is often derived from a monoclonalantibody, but other ligands (e.g., heregulin, cytokines) and receptors(e.g., NKp30) can also be used. The antigen-binding domain can includeany fragment of an antibody that retains antigen-binding function. Forexample, the CAR antigen-binding domain is often contributed by asingle-chain variable fragment (scFv), which is formed from the variableregions of heavy and light chains of a monoclonal antibody.

In one aspect, the transmembrane domain comprises a sequence of the zeta(ζ) chain associated with the T cell receptor complex, such as theintracellular domain of human CD3 ζ chain.

The intracellular stimulatory domain can include one or more of CD28,4-1BB (CD137), CD134 (OX-40), ICOS, and CD40L.

The antigen-binding domain, transmembrane domain, and the intracellularstimulatory domain(s) are linked either directly or via a spacersequence.

The CAR sequences are incorporated in an expression vector. Variousexpression vectors are known in the art and any such vector may beutilized. In some embodiments, the vector will be a retroviral orlentiviral vector. In other embodiments the vector will be derived fromadeno-associated virus.

Immune cells are transformed with the CAR and the CAR is then expressedon the cell surface. Typically, the immune cell stably expresses theCAR, although in some embodiments, the immune cell may transientlyexpress the CAR. The immune cell is thus transfected with a nucleicacid, e.g., mRNA, cDNA, DNA, encoding a CAR. Immune cells of thedisclosure include mammalian cells (e.g., human cells), and can beautologous cells, syngeneic cells, allogenic cells and even in somecases, xenogeneic cells, The cells are engineered to express a CAR and,therefore, are not found in nature. Exemplary immune cells include Tlymphocytes (T cells), natural killer (NK) cells, NKT cells, andmacrophages (including monocytes and dendritic cells).

The CAR-modified immune cells are then cultured to expand thepopulations obtain a suitable number of cells for a single dose or formultiple doses. In certain embodiments, one or more retinoid and/orrexinoid active agents are added to the expansion cultures during theculture period and have an effect on the CAR-modified cells directly.For example, in culturing CAR-MIC the one or more retinoid and/orrexinoid active agents added to the expansion cultures would be chosenfor their ability to, for example, suppress the development of Tregcells and/or their ability to promote the development Th17 cells. Insome embodiments, the one or more retinoid and/or rexinoid active agentsare included in the expansion culture of CAR-modified immune cells andadministered directly to a subject.

Immune Checkpoint Targeted Cancer Therapeutics

Immune checkpoint therapy targets regulatory pathways in thedifferentiation and activation of T cells to promote the passage of Tcell developmental program through these checkpoints so that anti-tumor(or other therapeutic) activity can be realized. The agents bringingabout immune checkpoint therapy are commonly called immune checkpointinhibitors and it should be understood that it is the check on T celldevelopment that is being inhibited. Thus, while many immune checkpointinhibitors also inhibit the interaction of receptor-ligand pairs (e.g.,anti-PD-1, anti-PD-L1, and CTLA-4), others (such as anti-OX40 andanti-ICOS) act as agonists of targets that release or otherwise inhibitthe check on T cell development, ultimately promoting effector functionand/or inhibiting regulatory function.

Disclosed herein is the use of retinoid and rexinoid receptor activemolecules (RAR/RXR active agents) as potentiators of the anti-tumoreffects of immune checkpoint inhibitor molecules in combination withCAR-modified immune cells. Molecules which inhibit immune checkpointproteins include antibodies which are specific to one or more of PD-1,PD-1 ligand, CTLA-4, TIM-3, LAG-3, B7-H3, and B7-H4.

Programed death-1 (PD-1) is a checkpoint protein on T cells and normallyacts as a type of “off switch” that helps keep the T cells fromattacking other cells in the body. It does this by binding to programmeddeath ligand-1 (PD-L1), a protein on some normal and cancer cells. WhenPD-1 binds to PD-L1, the T cells will not attack the target cells. Somecancer cells have large amounts of PD-L1, which helps them evade immuneattack. Monoclonal antibodies that target either PD-1 or PD-L1 can boostthe immune response against cancer cells and have shown a great deal ofpromise in treating certain cancers. Examples of monoclonal antibodiesthat target PD-1/PL-L1 include: the anti-PD-1 mAbs nivolumab (OPDIVO®,Bristol-Myers Squibb) and pembrolizumab (KEYTRUDA®, Merck & Co.),BMS-936559 (Bristol-Myers Squibb), pidilizumab (Medivation): and theanti-PD-L1 mAbs durvalumab (MEDI4736, IMFINZI™, Medimmune), atezolizumab(MPDL3280A; TECENTRIQ®, Hoffman-La Roche), avelumab (BAVENCIO®, EMDSerono). These antibodies have, variously, demonstrated utility intreating a variety of cancers including malignant melanoma (MM), renalcell carcinoma (RCC), Merkel cell carcinoma, urothelial carcinoma, andnon-small cell lung cancer (NSCLC). Non-antibody inhibitors ofPD-1/PD-I1 interaction are also being developed; for example, smallengineered proteins based on stefin A (called AFFIMER® molecules). Inaddition to PD-L1, PD-1 can also bind to PD-L2. In addition to PD-1,PD-L1 can also bind to B7-1 (CD80).

CTLA-4 is an immune checkpoint molecule expressed on the surface of CD4and CD8 T cells and on CD25+, FOXP3+T regulatory (Treg) cells. CTLA-4generates inhibitory signals that block T cell responses and enablestumor growth. Anti-CTLA-4 mAbs such as ipilimumab (YERVOY®;Bristol-Myers Squibb) cause shrinkage of tumors in animal models.Ipilimumab improves overall survival in MM patients and is approved forthe treatment of MM. Responses have been observed in RCC and NSCLC aswell. Other exemplary anti-CTLA-4 antibodies include tremelimumab(Medimmune).

The CTLA-4-blocking antibody ipilimumab gives durable responses only ina subset of melanoma patients and its effects on overall survival islimited. This has led to the search for resistance mechanisms to CTLA-4blockade and to the identification of the cytosolic enzyme indoleamine2,3-dioxygenase (IDO) as a potent mediator of melanoma resistance. IDOdirectly suppresses effector T cells and activates suppressive Tregcells thereby modulating the anti-tumor effects of CTLA-4 blockade.Inhibitors of IDO such as 1-methyl-tryptophan have T cell dependentanti-tumor effects and synergize with CTLA-4-blocking antibody tocontrol tumor growth and enhance survival.

TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) is amolecule selectively expressed on IFN-γ-producing CD4⁺ T helper 1 (Th1)and CD8⁺ T cytotoxic 1 (Tc1) T cells. TIM-3 is an immune checkpointreceptor that functions specifically to limit the duration and magnitudeof Th1 and Tc1 T-cell responses. Exemplary antibodies to TIM-3 aredisclosed in U.S. Patent Application Publication 20160075783 which isincorporated by reference herein for all it contains regardinganti-TIM-3 antibodies.

LAG-3 (lymphocyte-activation gene 3; CD223) negatively regulatescellular proliferation, activation, and homeostasis of T cells, in asimilar fashion to CTLA-4 and PD-1 and plays a role in Treg suppressivefunction. Exemplary antibodies to LAG-3 include GSK2831781(GlaxoSmithKline), BMS-986016 (Bristol-Myers Squibb) and the antibodiesdisclosed in U.S. Patent Application Publication 2011/0150892 which isincorporated by reference herein for all it contains regardinganti-LAG-3 antibodies.

The B7 family is a family of costimulatory proteins which are expressedon the surface of antigen-presenting cells and interact with ligands onT cells. B7-H3 (CD276) is one of the molecules in this family. Anantibody to B7-H3, enoblituzumab (EMPLICITI™, Bristol-Myers Squibb) isapproved for treatment of multiple myeloma. Another molecule in thefamily is B7-H4 (V-set domain-containing T-cell activation inhibitor 1),antibodies against which are in development.

Other immune checkpoint inhibitor targets, B- and T-cell attenuator(BTLA), inducible T-cell costimulator (ICOS), OX40 (tumor necrosisfactor receptor superfamily, member 4), and others are potentiallyuseful in the disclosed methods. Several anti-OX40 agonistic monoclonalantibodies are in early phase cancer clinical trials including MEDI0562and MEDI6469 (Medimmune), MOXR0916 (Genentech), and PF-04518600(Pfizer); as is an anti-ICOS agonistic antibody, JTX-2011 (JounceTherapeutics).

Disclosed herein are methods of potentiating the anti-cancer activity ofimmune checkpoint targeting immunotherapeutics including a CTLA-4inhibitor, a PD-1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, aPD-1 ligand (such as PDL-1), an inhibitor of a PD-1 ligand, an OX40agonist, an ICOS agonist, a B7-H3 protein, an inhibitor of a B7-H3protein, a B7-H4 protein, and an inhibitor of a B7-H4 protein. Incertain embodiments, the inhibitors are antibodies.

The immune checkpoint targeting immunotherapeutic antibodies can bewhole antibodies or antibody fragments. The terms “fragment of anantibody,” “antibody fragment,” and “functional fragment of an antibody”are used interchangeably herein to mean one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Theantibody fragment desirably comprises, for example, one or morecomplementary determining regions (CDRs), the variable region (orportions thereof), the constant region (or portions thereof), orcombinations thereof. Examples of antibody fragments include, but arenot limited to, a Fab fragment, which is a monovalent fragmentconsisting of the V_(L), V_(H), C_(L), and CH₁ domains; a F(ab′)₂fragment, which is a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a single chain Fv, in which the V_(L) and V_(H) domains arejoined by a peptide linker sequence; a Fab′ fragment, which results frombreaking the disulfide bridge of an F(ab′)₂ fragment using mild reducingconditions; a disulfide-stabilized Fv fragment (dsFv); and a domainantibody (dAb), which is an antibody single variable region domain (VHor VL) polypeptide that specifically binds antigen. It should also berealized that any of these forms of antigen-binding antibody fragmentscan provide the antigen binding domain of a CAR.

In alternative embodiments the antibody is replaced with another proteinthat similarly binds to the immune checkpoint target molecule. In someinstances these non-antibody molecules comprise an extracellular portionof the immune checkpoint target molecule's ligand or binding partner,that is, at least the extracellular portion needed to mediate binding tothe immune checkpoint target molecule. In some embodiments thisextracellular binding portion of the ligand is joined to additionalpolypeptide in a fusion protein. In some embodiments the additionalpolypeptide comprises an Fc or constant region of an antibody.

Methods of Treatment

Provided herein are methods of treating cancer in a mammal byadministering CAR-modified immune cells and one or more RAR/RXR activeagents. More specifically these are methods of cancer immunotherapy andmethods of potentiating CAR-modified immune cell immunotherapy. In someembodiments, immune checkpoint inhibitors are administered in additionto the CAR-modified immune cells and one or more RAR/RXR active agents.Also provided are methods of decreasing tumor burden, increasing thedisease-free survival in subject with cancer. Other embodiments relateto compositions comprising such agents for use in the treatment ofcancer, in cancer immunotherapy, and in potentiating CAR-modified immunecell-mediated immunotherapy. Still other embodiments relate tocompositions for use in making medicaments for the treatment of cancer,for cancer immunotherapy, and for potentiating CAR-modified immunecell-mediated immunotherapy. It is to be understood that the multipleagents used may be provided in separate compositions or medicamentswhich may be administered by separate routes of administration and/or atseparate times; nonetheless use of such multiple compositions ormedicaments is coordinated so that the patient to whom they areadministered receives the benefit of the combined, interacting activityof the multiple agents. For each method of treating cancer disclosedherein there are corresponding methods of cancer immunotherapy. For eachmethod of treating cancer or cancer immunotherapy there arecorresponding methods of potentiating cancer treatment/immunotherapy.

In some embodiments, the method comprises administering CAR-modifiedimmune cells and an RAR active agent. In some embodiments, the methodcomprises administering CAR-modified immune cells and an RARαantagonist. In some embodiments, the method comprises administeringCAR-modified immune cells and an RARγ agonist. In some embodiments, themethod comprises administering CAR-modified immune cells and two RARactive agents. In some embodiments, the method comprises administeringCAR-modified immune cells and an RARα antagonist an RAR agonist. In someembodiments, the method comprises administering CAR-modified immunecells and an RARα antagonist an RARγ selective agonist. In certainembodiments, the RARα antagonist is AGN194301, AGN193491, AGN193618,AGN194202, AGN194574, VTP196696, AGN19477, BMS185411, BMS614, Ro41-5253,or Ro46-5471. In some embodiments the RAR agonist is AGN190183,AGN190205, AFN204647, or tazarotene. In some embodiments, the RARγselective agonist is CD437, CD2325, CD666, or BMS961.

In some embodiments, the method comprises administering CAR-modifiedimmune cells and an RXR active agent. In some embodiments, the methodcomprises administering CAR-modified immune cells and an RXR antagonist.In some embodiments, the RXR antagonist is AGN195393 or LGN100849. Withrespect to the use of multiple RAR/RXR active agents in the various useor method of treatment embodiments described herein, any of thedisclosed general formula genera, sub-genera thereof, and individualspecies may be combined with any other general formula genera,sub-genera thereof, and individual species, each such combinationdefining an individual embodiment.

The compounds, pharmaceutical compositions, and methods disclosed hereinare particularly useful for the treatment of cancer. As used herein, theterm “cancer” refers to a cellular disorder characterized byuncontrolled or dysregulated cell proliferation, decreased cellulardifferentiation, inappropriate ability to invade surrounding tissue,and/or ability to establish new growth at ectopic sites. The term“cancer” includes, but is not limited to, solid tumors and hematologictumors. The term “cancer” encompasses diseases of skin, tissues, organs,bone, cartilage, blood, and vessels. The term “cancer” furtherencompasses primary and metastatic cancers. Included within the term“cancer cells” are cancer stem cells.

The disclosed methods can be used to treat any type of cancer known inthe art, such as, for example, melanoma, renal cell carcinoma, lungcancer, bladder cancer, breast cancer, cervical cancer, colon cancer,gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer,stomach cancer, salivary gland cancer, prostate cancer, pancreaticcancer, a hematologic cancer, or Merkel cell carcinoma. In someembodiments, the hematologic cancer is a leukemia, a lymphoma, amyelodysplastic syndrome, or a myeloma. In select embodiments aparticular type of cancer is treated. In other select embodiments aparticular type of cancer is excluded from treatment.

As used herein, the terms “treatment,” “treating,” and the like refer toobtaining a desired pharmacologic and/or physiologic effect. Preferably,the effect is therapeutic, i.e., the effect partially or completelycures a disease and/or adverse symptom attributable to the disease. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. The therapeutically effective amount may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the CAR-modified immune cells and oneor more retinoid and/or rexinoid active agents to elicit a desiredresponse in the individual. For example, a therapeutically effectiveamount of a retinoid-active agent disclosed herein is an amount whichpotentiates the anti-cancer activity of CAR-modified immune cells orleads to an increase in occurrence or duration of disease-free survivalin a subject.

Additionally, one or more retinoid and/or rexinoid active agents candecrease toxicity associated with CAR-modified immune cells by allowinga lower dose of CAR-modified immune cells to be administered with thesame efficacy or a higher dose of the CAR-modified immune cells can beadministered with the same degree of safety.

The term “treating” or “treatment” broadly includes any kind oftreatment activity, including the diagnosis, mitigation, or preventionof disease in man or other animals, or any activity that otherwiseaffects the structure or any function of the body of man or otheranimals. Treatment activity includes the administration of themedicaments, dosage forms, and pharmaceutical compositions describedherein to a patient, especially according to the various methods oftreatment disclosed herein, whether by a healthcare professional, thepatient his/herself, or any other person. Treatment activities includethe orders, instructions, and advice of healthcare professionals such asphysicians, physician's assistants, nurse practitioners, and the likethat are then acted upon by any other person including other healthcareprofessionals or the patient his/herself. In some embodiments, treatmentactivity can also include encouraging, inducing, or mandating that aparticular medicament, or combination thereof, be chosen for treatmentof a condition—and the medicament is actually used—by approvinginsurance coverage for the medicament, denying coverage for analternative medicament, including the medicament on, or excluding analternative medicament, from a drug formulary, or offering a financialincentive to use the medicament, as might be done by an insurancecompany or a pharmacy benefits management company, and the like. In someembodiments, treatment activity can also include encouraging, inducing,or mandating that a particular medicament be chosen for treatment of acondition—and the medicament is actually used—by a policy or practicestandard as might be established by a hospital, clinic, healthmaintenance organization, medical practice or physicians group, and thelike.

A typical dose of CAR-modified immune cells can be, for example, in therange of 1×10⁶ to 3×10¹⁰ cells per dose. In some embodiments,CAR-modified immune cells are administered at a dose of at least 1×10⁶cells/dose, at least 3×10⁶ cells/dose, at least 1×10⁷ cells/dose, atleast 3×10⁷ cells/dose, at least 1×10⁸ cells/dose, at least 3×10⁸cells/dose, at least 1×10⁹ cells/dose, at least 3×10⁹ cells/dose, atleast 1×10¹⁰ cells/dose, at least 3×10¹⁰ cells/dose, or a range definedby any two of the foregoing values. In some embodiments, the typicaldose of CAR-modified immune cells can be, for example, in the range of1×10⁵ to 1×10⁸ cells per kilogram of patient body weight. In someembodiments, CAR-modified immune cells are administered at a dose of atleast 1×10⁵ cells/kg, at least 3×10⁵ cells/kg, at least 6×10⁵ cells/kg,at least 1×10⁶ cells/kg, at least 3×10⁶ cells/kg, at least 6×10⁶cells/kg, at least 1×10⁷ cells/kg, at least 3×10⁷ cells/kg, or a rangedefined by any two of the foregoing values.

Therapeutic or prophylactic efficacy can be monitored by periodicassessment of treated patients. For repeated administrations overseveral days or longer, depending on the condition, the treatment can berepeated until a desired suppression of disease or disease symptomsoccurs. However, other dosage regimens may be useful and are within thescope of the present disclosure. The desired dosage can be delivered bya single bolus administration, by multiple bolus administrations, or bycontinuous infusion administration of the CAR-modified immune cells. Invarious embodiments the continuous infusion may extend for half an hour,for an hour, for several hours, for a day, or for several days.Treatment may comprise a single or multiple infusions.

In some embodiments, the CAR-modified immune cells are administered withother pre-treatment or simultaneous administrations of additionalagents. In some embodiments, subjects who are to receive CAR-modifiedimmune cells are pre-treated with a non-myeloablativelymphocyte-depleting regiment, such as, but not limited to, treatmentwith cyclophosphamide and/or fludarabine. In some embodiments,CAR-modified immune cells are administered with interleukin-2.

CAR-modified immune cells may be administered to a subject a single timeor multiple times. The cells can be administered weekly, biweekly,monthly, bimonthly, or upon evidence of cancer progression.

Depending on the type of cancer, and the patient to be treated, as wellas the route of administration, the disclosed RARα antagonists, RARγagonists and RXR antagonists may be administered at varyingtherapeutically effective doses to a patient in need thereof.

However, the dose administered to a mammal, particularly a human, in thecontext of the present methods, should be sufficient to effect atherapeutic response in the mammal over a reasonable timeframe. Oneskilled in the art will recognize that the selection of the exact doseand composition and the most appropriate delivery regimen will also beinfluenced by inter alia the pharmacological properties of theformulation, the nature and severity of the condition being treated, andthe physical condition and mental acuity of the recipient, as well asthe potency of the specific compound, the age, condition, body weight,sex and response of the patient to be treated, and the stage/severity ofthe disease.

Typical doses of RARα antagonists are 0.01 to 300 mg/m²/day; however,doses below or above this exemplary range are within the scope of thepresent disclosure. The daily dose can be about 0.5 to 100 mg/m²/day, 1to 90 mg/m²/day, 5 to 80 mg/m²/day; or at least 0.02, 0.03, 0.05, 0.07,0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 70 or100 mg/m²/day; or not more than 0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7,10, 15, 20, 25, 30, 50, 60, 70. 80, 90, 100, 125, 150, 175, 200, 225,250, 275, or 300 mg/m²/day; or a range defined by any two of theforegoing values.

Typical doses of RARγ agonists are 0.01 to 300 mg/m²/day; however, dosesbelow or above this exemplary range are within the scope of the presentdisclosure. The daily dose can be about 0.5 to 100 mg/m²/day, 1 to 90mg/m²/day, 5 to 80 mg/m²/day; or at least 0.02, 0.03, 0.05, 0.07, 0.1,0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 70 or 100mg/m²/day; or not more than 0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7, 10,15, 20, 25, 30, 50, 60, 70. 80, 90, 100, 125, 150, 175, 200, 225, 250,275, or 300 mg/m²/day; or a range defined by any two of the foregoingvalues.

Typical doses of RXR antagonists are 0.01 to 300 mg/m²/day; however,doses below or above this exemplary range are within the scope of thepresent disclosure. The daily dose can be about 0.5 to 100 mg/m²/day, 1to 90 mg/m²/day, 5 to 80 mg/m²/day; or at least 0.02, 0.03, 0.05, 0.07,0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 70 or100 mg/m²/day; or not more than 0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7,10, 15, 20, 25, 30, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225,250, 275, or 300 mg/m²/day; or a range defined by any two of theforegoing values.

The average surface area of a human body is generally accepted to be 1.9m² for an adult male, 1.6 m² for an adult female, and 1.33 m² for a12-13 year old child. These values can be used to calculate dose rangesfor daily dosage for the values in the preceding paragraphs. The totaldaily dosage of RAR/RXR active agents can be administered as a singledose or as two doses administered with a 24 hour period spaced 8 to 16,or 10 to 14, hours apart. The RAR/RXR active agents are administered incoordination with the CAR-modified immune cells and as above therapeuticor prophylactic efficacy can be monitored by periodic assessment oftreated patients. For repeated administrations over several days orlonger, depending on the condition, the treatment can be repeated untila desired suppression of disease or disease symptoms occurs. However,other dosage regimens may be useful and are within the scope of thedisclosure. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition.

The retinoid and/or rexinoid active agent can be administered to amammal using standard administration techniques, including parenteral,oral, intravenous, intraperitoneal, subcutaneous, pulmonary,transdermal, intramuscular, intranasal, buccal, sublingual, orsuppository administration. The term “parenteral,” as used herein,includes intravenous, intramuscular, subcutaneous, rectal, vaginal, andintraperitoneal administration. The CAR-modified immune cells areadministered to a mammal using peripheral systemic delivery byintravenous, intraperitoneal, or subcutaneous injection. The retinoidand/or rexinoid active agent preferably is suitable for oraladministration, for example as a pill, tablet or capsule.

Administration may be continuous or intermittent. The dosage may also bedetermined by the timing and frequency of administration. Thus, the RARαagonists disclosed herein can be given on a daily, weekly, biweekly, ormonthly basis for a period of time, followed by an optional drug holiday(drug free period) and that this drug administration/drug holiday cyclecan be repeated as necessary. In certain embodiments, the total dailydosage of RARα agonists can be administered as a single dose or as twodoses administered with a 24 hour period spaced 8 to 16, or 10 to 14,hours apart.

The CAR-modified immune cells and retinoid and/or rexinoid active agentsdisclosed herein may be administered at substantially the same time(within 1 hr. of each other) or at different times. In some embodiments,the subject is pre-treated with a retinoid and/or rexinoid active agentat least 30 min, at least 1 hr., or at least 2 hrs. beforeadministration of the CAR-modified immune cells. In preferredembodiments, the subject is pretreated with a retinoid and/or rexinoidactive agent for at least 12 hours, or 1 day, 2, 3, 4, 5 days prior toadministration of the CAR-modified immune cells. In some embodiments,the subject is pretreated with a retinoid and/or rexinoid active agentfor 5-10 days, for example 6, 7, or 8 days, prior to administration ofthe CAR-modified immune cells; or for any range defined by any of twothe foregoing values. In some embodiments, the retinoid and/or rexinoidactive agent is administered after the onset of CAR-modified immunecells administration, for example, the same day, the next day, two dayslater, three days later, a week later, etc. It is anticipated thatRAR/RXR therapy will be administered on a daily basis for a period oftime and may be given longer than CAR-modified immune cells. In someembodiments administration the RAR and/or RXR active agent(s) continuesuntil such time as the patient has demonstrated a durable completeresponse (that is, a complete response for at least 6 months followingadministration of the CAR-modified immune cells). In other embodiments,administration of the RAR and/or RXR active agent(s) continues for aslong as tumor regression proceeds or there is stable disease.

The CAR-modified immune cells and retinoid and/or rexinoid active agentsdisclosed herein may be administered in combination with other drugs,such as at least one other anticancer agent including, for example, anychemotherapeutic agent known in the art, ionization radiation, smallmolecule anticancer agents, cancer vaccines, biological therapies (e.g.,other monoclonal antibodies, cancer-killing viruses, gene therapy, andadoptive T-cell transfer), and/or surgery. In other embodiments theCAR-modified immune cells and retinoid and/or rexinoid active agents arethe only therapeutic reagents administered or the only treatment given;or the only treatment or reagents given, the primary utility of which isto promote an anti-cancer immune response.

The effectiveness of cancer therapy is typically measured in terms of“response.” The techniques to monitor responses can be similar to thetests used to diagnose cancer such as, but not limited to:

-   -   A lump or tumor involving some lymph nodes can be felt and        measured externally by physical examination.    -   Some internal cancer tumors will show up on an x-ray or CT scan        and can be measured with a ruler.    -   Blood tests, including those that measure organ function can be        performed.    -   A tumor marker test can be done for certain cancers.

Regardless of the test used, whether blood test, cell count, or tumormarker test, it is repeated at specific intervals so that the resultscan be compared to earlier tests of the same type.

Response to cancer treatment is defined several ways:

-   -   Complete response—all of the cancer or tumor disappears; there        is no evidence of disease. Expression level of tumor marker (if        applicable) may fall within the normal range.    -   Partial response—the cancer has shrunk by a percentage but        disease remains. Levels of a tumor marker (if applicable) may        have fallen (or increased, based on the tumor marker, as an        indication of decreased tumor burden) but evidence of disease        remains.    -   Stable disease—the cancer has neither grown nor shrunk; the        amount of disease has not changed. A tumor marker (if        applicable) has not changed significantly.    -   Disease progression—the cancer has grown; there is more disease        now than before treatment. A tumor marker test (if applicable)        shows that a tumor marker has risen.

Other measures of the efficacy of cancer treatment include intervals ofoverall survival (that is time to death from any cause, measured fromdiagnosis or from initiation of the treatment being evaluated)),cancer-free survival (that is, the length of time after a completeresponse cancer remains undetectable), and progression-free survival(that is, the length of time after disease stabilization or partialresponse that resumed tumor growth is not detectable).

There are two standard methods for the evaluation of solid cancertreatment response with regard to tumor size (tumor burden), the WHO andRECIST standards. These methods measure a solid tumor to compare acurrent tumor with past measurements or to compare changes with futuremeasurements and to make changes in a treatment regimen. In the WHOmethod, the solid tumor's long and short axes are measured with theproduct of these two measurements is then calculated; if there aremultiple solid tumors, the sum of all the products is calculated. In theRECIST method, only the long axis is measured. If there are multiplesolid tumors, the sum of all the long axes measurements is calculated.However, with lymph nodes, the short axis is measured instead of thelong axis.

In some embodiments of the current method, the tumor burden of a treatedpatient is reduced by about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%about 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about95%, about 100%, or any range bound by these values.

In other embodiments, the 1-year survival rate of treated subjects isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55% about 60%,about 65%, about 70%, about 75%, about 80%, about 90%, about 95%, about100%, or any range bound by these values.

In other embodiments, the 5-year survival rate of treated subjects isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55% about 60%,about 65%, about 70%, about 75%, about 80%, about 90%, about 95%, about100%, or any range bound by these values.

In other embodiments, the 10-year survival rate of treated subjects isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55% about 60%,about 65%, about 70%, about 75%, about 80%, about 90%, about 95%, about100%, or any range bound by these values.

In yet other embodiments, the subject has a sustained remission of atleast 6 months, at least 7 months, at least 8 months, at least 9 months,at least 10 months, at least 11 months, at least 12 months, at least 14months, at least 16 months, at least 18 months, at least 20 months, atleast 22 months, at least 24 months, at least 27 months, at least 30months, at least 33 months, at least 36 months, at least 42 months, atleast 48 months, at least 54 months, or at least 60 months or more.

In other embodiments, the method may help to treat or alleviateconditions, symptoms, or disorders related to cancer. In someembodiments, these conditions or symptoms may include, but are notlimited to, anemia, asthenia, cachexia, Cushing's Syndrome, fatigue,gout, gum disease, hematuria, hypercalcemia, hypothyroidism, internalbleeding, hair loss, mesothelioma, nausea, night sweats, neutropenia,paraneoplastic syndromes, pleuritis, polymyalgia rheumatica,rhabdomyolysis, stress, swollen lymph nodes, thrombocytopenia, Vitamin Ddeficiency, or weight loss. In other embodiments, the administration ofboth the RARα agonist and CAR-modified immune cells prolongs thesurvival of the individual being treated relative to treatment with theCAR-modified immune cells alone.

LIST OF PARTICULAR EMBODIMENTS

The following listing of embodiments is illustrative of the variety ofembodiments with respect to breadth, combinations and sub-combinations,class of invention, etc., elucidated herein, but is not intended to bean exhaustive enumeration of all embodiments finding support herein.

Embodiment 1

A method of cancer immunotherapy comprising administering to a subjectin need thereof chimeric antigen receptor-modified immune cells(CAR-MIC) and at least one retinoid active agent and/or rexinoid activeagent (RAR/RXR active agent).

Embodiment 2

A method of treating cancer comprising administering to a subject inneed thereof (CAR-MIC) and at least one RAR/RXR active agent.

Embodiment 3

A method of potentiating CAR-MIC cancer immunotherapy comprisingadministering at least one RAR/RXR active agent to a cancer patient whois receiving, has received, or is scheduled to receive CAR-MIC.

Embodiment 4

A method of cancer immunotherapy comprising administering to a subjectin need thereof CAR-MIC, wherein the CAR-MIC are cultured in a culturemedium comprising at least one RAR/RXR active agent prior to beingadministered to the subject.

Embodiment 5

A method of prolonging the disease-free survival of a cancer patientcomprising administering CAR-MIC and at least one RAR/RXR active agent.

Embodiment 6

A method of decreasing toxicity of CAR-MIC comprising administering to asubject in need thereof at least one RAR/RXR active agent in combinationwith the CAR-MIC such that as a result of the combination, a lower doseof CAR-MIC are administered with greater safety and equal efficacy thanif the CAR-MIC were administered alone; or alternatively allowingequally safe administration of a higher dose of CAR-MIC with greaterefficacy.

Embodiment 7

A method of expanding the number of CAR-MIC in vitro comprisingculturing the CAR-MIC in a culture medium comprising at least oneRAR/RXR active agent.

Embodiment 8

The method of any one of Embodiments 1-6, wherein the CAR-MIC arecultured in a culture medium comprising at least one RAR/RXR activeagent prior to being administered to the subject.

Embodiment 9

The method of any one of Embodiments 1-8, wherein the at least oneRAR/RXR active agent is a RARα antagonist, a RARγ agonist, a RXRantagonist, or a combination thereof.

Embodiment 10

The method of any one of Embodiments 1-6 or 8-9, further comprisingadministration of an immune checkpoint inhibitor.

Embodiment 11

The method of Embodiment 10 wherein the immune checkpoint inhibitor isan inhibitor of at least one of CTLA-4, PD-1, TIM-3, LAG-3, PD-L1ligand, B7-H3, B7-H4, BTLA, or is an ICOS, or OX40 agonist.

Embodiment 12

The method of Embodiment 10 or 11, wherein the immune checkpointinhibitor is an antibody.

Embodiment 13

The method of any one of Embodiments 1-12, wherein the at least oneRAR/RXR active agent comprises a Retinoic Acid Receptor (RAR) activeagent.

Embodiment 14

The method of Embodiment 13 wherein the at least one RAR/RXR activeagent is a RAR active agent.

Embodiment 15

The method of Embodiment 13 or 14, wherein the RAR active agent is aRARα antagonist.

Embodiment 16

The method of Embodiment 15, wherein the RAR active agent is a selectiveRARα antagonist.

Embodiment 17

The method of Embodiment 13 or 14, wherein the RAR active agent is aRARγ agonist.

Embodiment 18

The method of Embodiment 17, wherein the RAR active agent is a selectiveRARγ agonist.

Embodiment 19

The method of any one of Embodiments 1-13, wherein the at least oneRAR/RXR active agent comprises a Retinoid X Receptor (RXR) active agent.

Embodiment 20

The method of Embodiment 19, wherein the at least one RAR/RXR agent is aRetinoid X Receptor (RXR) active agent.

Embodiment 21

The method of embodiment 20, wherein the RXR active agent is a RXRantagonist.

Embodiment 22

The method of any one of Embodiments 1-21, wherein the at least oneRAR/RXR active agent comprises at least two RAR active agents.

Embodiment 23

The method of Embodiment 22, wherein a first RAR active agent is a RARαantagonist, and a second RAR active agent is a RARγ selective agonist.

Embodiment 24

The method of Embodiment 22, wherein a first RAR active agent is a RARαselective antagonist, and a second RAR active agent is a RARγ agonist.

Embodiment 25

The method of Embodiment 22, wherein a first RAR active agent is a RARαselective antagonist, and a second RAR active agent is a RARγ selectiveagonist.

Embodiment 26

The method of any one of Embodiments 9, 15-16, or 23-25, wherein theRARα antagonist is a compound of general formula (I)

wherein R¹, R², R³, and R⁶ are independently H or C₁₋₆ alkyl; R⁴ and R⁵are independently H or F; Ar is phenyl, pyridyl, thienyl, furyl, ornaphthyl; X is C(CH₃)₂, O, S, or NR⁷, wherein R⁷ is H or C₁₋₆ alkyl; X¹is H or halogen such as F, Cl or Br; and R⁸ is H or OH.

Embodiment 27

The method of Embodiment 26, wherein the RARα antagonist is:

Embodiment 28

The method of any one of Embodiments 9, 15-16, or 23-25, wherein theRARα antagonist is a compound of general formula (II)

wherein R¹ and R² are independently C₁₋₆ alkyl; X is O, S, or CH₂; Y isO, S, CH₂, or NR³, wherein R³ is C₁₋₆ alkyl; Z is Cl or Br; W is H orOH; and U is independently H or F.

Embodiment 29

The method of Embodiment 28, wherein the RARα antagonist is:

Embodiment 30

The method of any one of Embodiments 9, 15-16, or 23-25, wherein theRARα antagonist is a compound of general formula (III)

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; Ar isphenyl, pyridyl, thienyl, furyl, or naphthyl; X is O, S, N, or CH₂; W isH or OH; and Z is Cl or Br.

Embodiment 31

The method of any one of Embodiments 9, 15-16, or 23-25, wherein theRARα antagonist is BMS185411, BMS614, Ro41-5253, Ro46-5471, or

Embodiment 32

The method of any one of Embodiments 9, 15-16, or 23-25, wherein theRARγ agonist is a RARγ agonist of general formula IV

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; and Xis O, S, CH₂, C(R⁴)₂, or NR⁵, wherein R⁴ and R⁵ are independently H orC₁₋₆ alkyl.

Embodiment 33

The method of any one of Embodiments 9, 15-16, or 23-25, wherein theRARγ agonist is:

Embodiment 34

The method of any one of Embodiments 9, 15-16, or 23-25, wherein theRARγ agonist is a selective RARγ agonist selected from CD437, CD2325,CD666, and BMS961.

Embodiment 35

The method of any one of Embodiments 9 or 19-20, wherein the RXRantagonist is selected from

Embodiment 36

The method of any one of Embodiments 9 or 19-20, wherein the RXRantagonist is AGN195393, or LGN100849.

Embodiment 37

The method of any one of Embodiments 1-6 or 8-36, further comprisingadministering at least one cancer chemotherapy agent.

Embodiment 38

The method of any one of Embodiments 1-6 or 8-37, wherein the subject orpatient is pretreated with the at least one RAR/RXR active agent priorto administration of the CAR-MIC.

Embodiment 39

The method of Embodiment 38 wherein the at least one RAR/RXR activeagent is administered at least 12 hours before administration of theCAR-MIC.

Embodiment 40

The method of Embodiment 39 wherein the at least one RAR/RXR activeagent is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 daysbefore administration of the CAR-MIC.

Embodiment 41

The method of any one of Embodiments 1-6 or 8-40 wherein the subject orpatient is treated with the at least one RAR/RXR active agent concurrentwith or subsequent to administration of the CAR-MIC.

Embodiment 42

The method of Embodiment 41, wherein treatment (as distinct frompretreatment, if any) commences on the same day as administration orfirst administration of the CAR-MIC.

Embodiment 43

The method of Embodiment 41, wherein treatment (as distinct frompretreatment, if any) commences 1, 2, 3, 4, 5, 6, or 7 days afteradministration or first administration of the CAR-MIC.

Embodiment 44

The method of any one of Embodiments 41-43, wherein treatment with theat least one RAR/RXR active agent continues for at least 6 monthsfollowing administration or 1st administration of the CAR-MIC.

Embodiment 45

The method of any one of Embodiments 41-43, wherein treatment with theat least one RAR/RXR active agent continues until a durable completeresponse is obtained.

Embodiment 46

The method of any one of Embodiments 41-43, wherein treatment with theat least one RAR/RXR active agent continues as long as there iscontinued tumor regression.

Embodiment 47

The method of any one of Embodiments 41-43, wherein treatment with theat least one RAR/RXR active agent continues as long as there is stabledisease or the cancer does not progress.

Embodiment 48

The method of any one of Embodiments 1-6 or 8-47, wherein the at leastone RAR/RXR active agent is administered daily.

Embodiment 49

The method of any one of Embodiments 1-48, wherein the CAR-MIC is aCAR-T cell.

Embodiment 50

The method of any one of Embodiments 1-48, wherein the CAR-MIC is aCAR-NKT cells

Embodiment 51

The method of any one of Embodiments 1-48, wherein the CAR-MIC is aCAR-NK cell.

Embodiment 52

The method of any one of Embodiments 1-48, wherein the CAR-MIC is aCAR-macrophage.

Embodiment 53

One or more RAR/RXR active agents for use in cancer immunotherapy in apatient who is receiving, has received, or is scheduled to receiveCAR-MIC, whereby the immunotherapeutic effect of the CAR-MIC ispotentiated.

Embodiment 54

CAR-MIC and at least one RAR/RXR active agent for use in cancerimmunotherapy.

Embodiment 55

CAR-MIC and at least one RAR/RXR active agent for use in prolonging thedisease-free survival of a cancer patient.

Embodiment 56

One or more RAR/RXR active agents for use in reducing the toxicity ofCAR-MIC therapy.

Embodiment 57

CAR-MIC and at least one RAR/RXR active agent for use in treatingcancer.

Embodiment 58

Use of one or more RAR/RXR active agents in the manufacture of amedicament for potentiating the immunotherapeutic effect of CAR-MIC inthe treatment of cancer.

Embodiment 59

Use of CAR-MIC and at least one RAR/RXR active agent in the manufactureof a medicament for cancer immunotherapy.

Embodiment 60

Use of CAR-MIC and at least one RAR/RXR active agent in the manufactureof a medicament for prolonging the disease-free survival of a cancerpatient.

Embodiment 61

Use of one or more RAR/RXR active agents in the manufacture of amedicament for reducing the toxicity of CAR-MIC therapy.

Embodiment 62

Use of CAR-MIC and at least one RAR/RXR active agent in the manufactureof a medicament for treating cancer. It should be manifest that each ofEmbodiments 53-62 can be modified in a manner similar to themodification of Embodiments 1-6 by Embodiments 8-52.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification.

Example 1 RARα Signaling Induces Foxp3 Expression

It is important to determine which of the RAR (RARα, RARβ, RARγ)signaling pathways is important in the induction of Foxp3 expression. Todetermine this, naive CD4⁺ CD25⁻ FoxP3⁻ cells were purified from aFoxp3-GFP mouse using flow cytometry by sorting and isolating based upona GFP⁻ phenotype. These cells were activated polyclonally with aCD3 invitro in the presence of IL-2 and TGF-β. To identify the RAR involved inRA-induced Foxp3 expression, the cultured cells were incubated with RARselective agonists. The cultured cells were then scored for thefrequency of GFP⁺ (Foxp3⁺). Wth respect to the use of selectiveagonists, only the RARα agonist exerted significant impact on theexpression of Foxp3 inducing nearly 100% Foxp3+ T cells, withenhancement on the expression of α4β7 and CCR9 (gut homing receptors)(FIG. 1). The RARγ and RARβ agonists were without effect. These resultsindicate that RARα selective agonists could be useful in reducing asymptom of inflammation or an autoimmune disorder. Conversely, RARαselective antagonists or inverse agonists could be useful todownregulate the production of immunosuppressive Treg cells therebypromoting an immune response, such as an anti-cancer immune response.

Example 2 Binding of Test Compounds to RAR and RXR Receptors andActivation of Reporter Genes

Retinoic acid receptor transactivation activity and binding efficienciesare determined essentially as described in U.S. Pat. Nos. 5,298,429 and5,071,773, incorporated by reference herein. Transactivation assaysemploy expression plasmids encoding the full length receptors RARα,RARβ, RARγ, RXRα, RXRβ, and RXRγ. Reporter plasmids containing theherpes virus thymidine kinase promoter and the appropriate retinoic acidreceptor response element (RAREs) or retinoid X receptor responseelement (RXREs) are positioned upstream of an open coding regionencoding firefly luciferase.

Binding assays are performed using a classic competition assay format inwhich cloned receptor RAR and RXR molecules are first loaded with eitherradiolabeled all-trans-retinoic acid (RAR) or radiolabeled 9-cisretinoic acid (RXR), and then the amount of radioactivity liberated withincreasing concentration of test compound is measured.

The assays are used to identify RARα selective antagonists, RARγselective agonists and RXR selective antagonists as disclosed hereinabove.

Example 3 Pharmacological Activation of RARγ Signaling Using RARγAgonists has a Cooperative Effect with Anti-CTLA-4 Antibody in Rejectionof B 16 Melanoma Cells

The anti-tumor effects of anti-CTLA-4 antibody treatment combined with10 nM RARγ agonist (AGN204647 (IRX4647)) are examined in C₅₇BL/6 miceengrafted with B16F10 tumor cells. Mice treated with vehicles only donot show a survival advantage (0%) over untreated control mice. Thesurvival rate of the mice treated with anti-CTLA-4 antibody alone is 40%at 50 days while the mice treated with RARγ agonist alone have a 30%survival in the same time. Remarkably, mice treated with bothanti-CTLA-4 antibody and RARγ agonist have a 100% survival at 50 daysindicating that these two agents cooperate to eliminate the B16 melanomacells. Surviving mice that undergo combination treatment are resistantto re-challenge with twice the dose of live tumor cells indicating theeffective formation of B16-specific memory cells. Importantly, theanti-melanoma effect is obtained with this combination of drugs withoutsigns of acute or delayed toxicity

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used hereinthe terms “about” and “approximately” means within 10 to 15%, preferablywithin 5 to 10%. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof the invention are approximations, the numerical values set forth inthe specific examples are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in their respective testingmeasurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. A method of cancer immunotherapy comprising administering chimericantigen receptor-modified immune cells (CAR-MIC) and retinoid and/orrexinoid means for modulating Treg and/or Th17 cells (immunomodulatorymeans).
 2. The method of claim 1, wherein the CAR-MIC have been culturedin a culture medium comprising at least one immunomodulatory means priorto administration of the CAR-MIC to the cancer patient.
 3. The method ofclaim 1, further comprising administration of an immune checkpointinhibitor that is an inhibitor of at least one of CTLA-4, PD-1, TIM-3,LAG-3, PD-L1 ligand, B7-H3, B7-H4, BTLA, or is an ICOS, or OX40 agonist,wherein the immune checkpoint inhibitor is an antibody.
 4. The method ofclaim 1, wherein the immunomodulatory means comprises at least two RARactive agents.
 5. The method of claim 4, wherein a firstimmunomodulatory means is a RARα antagonist, and a secondimmunomodulatory means is a RARγ selective agonist.
 6. The method ofclaim 4, wherein a first immunomodulatory means is a RARα selectiveantagonist, and a second immunomodulatory means is a RARγ agonist. 7.The method of claim 1, wherein the immunomodulatory means is not a RARαantagonist compound of general formula (I)

wherein R¹, R², R³, and R⁶ are independently H or C₁₋₆ alkyl; R⁴ and R⁵are independently H or F; Ar is phenyl, pyridyl, thienyl, furyl, ornaphthyl; X is C(CH₃)₂, O, S, or NR⁷, wherein R⁷ is H or C₁₋₆ alkyl; X¹is H or halogen such as F, Cl or Br; and R⁸ is H or OH.
 8. (canceled) 9.The method of claim 1, wherein the immunomodulatory means is not a RARαantagonist compound of general formula (II)

wherein R¹ and R² are independently C₁₋₆ alkyl; X is O, S, or CH₂; Y isO, S, CH₂, or NR³, wherein R³ is C₁₋₆ alkyl; Z is Cl or Br; W is H orOH; and U is independently H or F.
 10. (canceled)
 11. The method ofclaim 1, wherein the immunomodulatory means is not a RARα antagonistcompound of general formula (III):

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; Ar isphenyl, pyridyl, thienyl, furyl, or naphthyl; X is O, S, N, or CH₂; W isH or OH; and Z is Cl or Br.
 12. The method of claim 1, wherein theimmunomodulatory means is not BMS185411, BMS614, Ro41-5253, Ro46-5471,or


13. The method of claim 1, wherein the immunomodulatory means is not:


14. The method of claim 1, wherein the immunomodulatory means is not aRARγ agonist of general formula (IV):

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; and Xis O, S, CH₂, C(R⁴)₂, or NR⁵, wherein R⁴ and R⁵ are independently H orC₁₋₆ alkyl.
 15. The method of claim 1, wherein the immunomodulatorymeans is not a RARγ agonist selected from CD437, CD2325, CD666, andBMS961.
 16. The method of claim 1, wherein the immunomodulatory means isnot a RXR antagonist selected from AGN195393, LGN100849,


17. The method of claim 1, wherein the CAR-MIC is a CAR-T cell.
 18. Themethod of claim 1, comprising administering the immunomodulatory meansfor a period of 5 to 10 days prior to administration of CAR-MIC.
 19. Themethod of claim 1, comprising administering the immunomodulatory meanssubsequent to administration of the CAR-MIC.
 20. The method of claim 1,comprising administering the immunomodulatory means concurrent withadministration of the CAR-MIC.